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Farm animals are a potential reservoir for human Clostridium difficile infection (CDI), particularly PCR ribotype 078 which is frequently found in animals and humans. Here, whole genome single-nucleotide polymorphism (SNP) analysis was used to study the evolutionary relatedness of C. difficile 078 isolated from humans and animals on Dutch pig farms. All sequenced genomes were surveyed for potential antimicrobial resistance determinants and linked to an antimicrobial resistance phenotype. We sequenced the whole genome of 65 C. difficile 078 isolates collected between 2002 and 2011 from pigs (n?=?19), asymptomatic farmers (n?=?15) and hospitalised patients (n?=?31) in the Netherlands. The collection included 12 pairs of human and pig isolates from 2011 collected at 12 different pig farms. A mutation rate of 1.1 SNPs per genome per year was determined for C. difficile 078. Importantly, we demonstrate that farmers and pigs were colonised with identical (no SNP differences) and nearly identical (less than two SNP differences) C. difficile clones. Identical tetracycline and streptomycin resistance determinants were present in human and animal C. difficile 078 isolates. Our observation that farmers and pigs share identical C. difficile strains suggests transmission between these populations, although we cannot exclude the possibility of transmission from a common environmental source.

It has been reported that low-pH-induced fusion of influenza virus with liposomes results in rapid and extensive release of both low- and high-molecular-weight substances from the liposomes [Gunther-Ausborn et al., J. Biol. Chem. 270 (1995) 29279-29285; Shangguan et al., Biochemistry 35 (1996) 4956-4965]. Here, we demonstrate retention of encapsulated water-soluble compounds during fusion of Semliki Forest virus (SFV) or Sindbis virus with liposomes at low pH. Under conditions allowing complete fusion of the liposomes, a limited fluorescence dequenching of liposome-encapsulated calcein was observed, particularly for SFV. Also, radioactively labeled inulin or sucrose were largely retained. Freezing and thawing of the viruses in the absence of sucrose resulted in an enhanced leakiness of fusion. These results support the notion that the alphavirus fusion event per se is non-leaky and may well involve a discrete hemifusion intermediate. (C) 2002 Published by Elsevier Science B.V. on behalf of the Federation of European Biochemical Societies.

Semliki Forest virus (SFV) has been shown previously to fuse efficiently with cholesterol-and sphingolipid-containing liposomal model membranes in a low-pH-dependent manner. Several steps can be distinguished in this process, including low-pH-induced irreversible binding of the virus to the liposomes, facilitated by target membrane cholesterol, and subsequent fusion of the viral membrane with the liposomal bilayer, specifically catalyzed by target membrane sphingolipid. Binding and fusion are mediated by the heterodimeric viral envelope glycoprotein E2/E1. At low pH the heterodimer dissociates, and the El monomers convert to a homotrimeric structure, the presumed fusion-active conformation of the viral spike. In this paper, we demonstrate that SFV-liposome fusion is specifically inhibited by Zn2+ ions. The inhibition is al the level of the fusion reaction itself, since virus-liposome binding was found to be unaffected. Zn2+ did not inhibit E2/E1 dissociation, but severely inhibited exposure of an acid-specific epitope on El, El homotrimer formation, and acquisition of trypsin-resistance. It is concluded that virus-liposome binding solely requires low-pH-induced E2/E1 heterodimer dissociation, while fusion depends on further rearrangements in the El spike protein. As these rearrangements occur subsequent to the binding step, their precise course, including the formation of a fusion complex, may be influenced by interaction of El with target membrane lipids. (C) 1997 Academic Press.

Anionic charge-modified human serum albumin (HSA) has previously been shown to exert potent in vitro activity against human immunodeficiency virus type 1 (HIV-1). In these studies, introduction of the additional negative charges was performed by derivatizing the epsilon-amino groups of lysine residues with succinic (Suc-HSA) or cis-aconitic anhydride (Aco-HSA), by which primary amino groups are replaced with carboxylic acids. The anti-HIV-1 activity was related to inhibition of gp41-mediated membrane fusion. Here, we investigated the activity of aconitylated and succinylated proteins on influenza Virus membrane fusion, which is mediated by the viral membrane glycoprotein hemagglutinin (HA). Aco-HSA and Suc-HSA markedly inhibited the rates and extents of fusion of fluorescently labeled virosomes bearing influenza HA, with target membranes derived from erythrocytes. The inhibitory activity was dependent on the overall negative-charge density; HSA modified with 36 or less extra negative charges failed to inhibit fusion. The inhibition of fusion showed a certain degree of specificity for the protein carrying the negative charges: polyanionic HSA and beta-lactoglobulin A derivatives had fusion-inhibitory activity, whereas succinylated BSA, lactalbumin, lactoferrin, lysozyme, and transferrin were inactive. Aco(60)-HSA and Aco-beta-lactoglobulin A inhibited influenza virus membrane fusion in a concentration-dependent manner, IC50 values being about 4 and 10 mu g/mL, respectively. HA-mediated membrane fusion is pH dependent. Aco(60)-HSA did not induce a shift in the pH threshold or in the pH optimum. Fusion with liposomes of another low pH-dependent virus, Semliki Forest virus, was not specifically affected by any of the compounds reported here. In view of some structural and functional similarities between influenza HA and the HIV-1 gp120/gp41 complex, it is tempting to postulate that the current results might have some implications for the anti-HIV-1 mechanism of polyanionic proteins. (C) 1997 Elsevier Science Inc.

The alphavirus Semliki Forest virus (SFV) enters cells through receptor-mediated endocytosis. Subsequently, triggered by the acid pH in endosomes, the viral envelope fuses with the endosomal membrane. Membrane fusion of SFV has been shown previously to be dependent on the presence of cholesterol in the target membrane. Recently, we have demonstrated that fusion of SFV also requires sphingolipids [Nieva, J. L., Bron, R., Corver, J., & Wilschut, J. (1994) EMBO J. 13, 2797-2804]. In the present paper, we show that the activation of low-pH-dependent fusion of SFV by sphingolipids is a stereospecific process. Pyrene-labeled SFV fused rapidly and extensively with liposomes consisting of a mixture of phosphatidylcholine, phosphatidylethanolamine, and cholesterol, supplemented with low concentrations of D-erythro-ceramide, representing the naturally occurring sphingolipid stereoisomer. Fusion was assessed by a decrease in the pyrene excimer fluorescence. L-erythro-, D-threo-, and L-threo-ceramide did not support fusion of the virus. Similar results were obtained with the corresponding sphingomyelin stereoisomers, The stereospecificity of SFV fusion activation was confirmed by using an assay based on degradation of the viral capsid protein by trypsin encapsulated in the target liposomes. Fusion mediated by D-erythro-ceramide was not affected by the additional presence in the target liposomes of ceramide stereoisomers incapable of fusion activation. Binding of the virus to the liposomes, as assessed by flotation on sucrose density gradients, was not dependent on the presence of fusion-competent or fusion-incompetent sphingolipids in the liposomes, The results of this study support the notion that a stereospecific interaction of the viral fusion protein with D-erythro sphingolipids in the target membrane represents an essential step in the activation of the fusion capacity of SFV.

Low-pH-induced membrane fusion of Semliki Forest virus (SFV) in a model system is mediated by sphingolipids in the target membrane; ceramide is the sphingolipid minimally required (J. L. Nieva, R. Bron, J. Corver, and J. Wilschut, EMBO J. 13:2797-2804, 1994). Here, using various ceramide analogs, we demonstrate that sphingolipid-dependent fusion of SFV with cholesterol-containing liposomes exhibits remarkable molecular specificity, the 3-hydroxyl group and the 4,5-trans carbon-carbon double bond of the sphingosine backbone being critical for the sphingolipid to mediate the process. This observation supports the notion that sphingolipids act as a cofactor in SFV fusion, interacting directly with the viral fusion protein to induce its ultimate fusion-active conformation.

Enveloped animal viruses, such as Semliki Forest virus (SFV), utilize a membrane fusion strategy to deposit their genome into the cytosol of the host cell. SFV enters cells through receptor-mediated endocytosis, fusion of the viral envelope occurring subsequently from within acidic endosomes. Fusion of SFV has been demonstrated before to be strictly dependent on the presence of cholesterol in the target membrane. Here, utilizing a variety of membrane fusion assays, including an on-line fluorescence assay involving pyrene-labeled virus, we demonstrate that low-pa-induced fusion of SFV with cholesterol-containing liposomal model membranes requires the presence of sphingomyelin or other sphingolipids in the target membrane. The minimal molecular characteristics essential for supporting SFV fusion are encompassed by a ceramide. The action of the sphingolipids is confined to the actual fusion event, cholesterol being necessary and sufficient for low-pH-dependent binding of the virus to target membranes. Complex formation of the sphingolipids with cholesterol is unlikely to be important for the induction of SFV-liposome fusion, as sphingolipids that do not interact appreciably with cholesterol, such as galactosylceramide, effectively support the process. The remarkably low levels of sphingomyelin required for half-maximal fusion (1-2 mole%) suggest that sphingolipids do not play a structural role in the SFV fusion process, but rather act as a cofactor, possibly activating the viral fusion protein in a specific manner.

This thesis describes the application of molecular typing methods to study the epidemiology and evolution of Clostridium difficile. C. difficile is a gut bacterium that is a major cause of antibiotic associated diahrrea in the developed world. The emergence of the __hypervirulent__ types causing C. difficile infections (CDI) with more severe complications has driven the demand for rapid molecular assays that can detect these types. We aimed to identify novel molecular markers for __hypervirulent__ C. difficile and use these markers to develop new rapid assays. This thesis describes the development and evaluation of a new (multiplex) real-time PCR assay to diagnose CDI. It also provides an overview of the currently applied methods to study CDI. We report on an internationally-standardized protocol for high resolution, capillary gel-based electrophoresis, PCR-ribotyping. Furthermore, we describe two novel genetic markers that are linked to two C. difficile phylogenetic lineages. We describe the application of multilocus sequence typing to study the population structure of C. difficile. We report on whole genome sequencing (WGS) and the related single nucleotide polymorphism (SNP) analysis, a recently emerged typing method that we used to study transmission of C. difficile type 078 between animals (pigs) and humans (farmers).

Over the last years major advances have been made in the field of C. difficile research. Despite the continuous progress of research in C. difficile epidemiology and molecular biology. This thesis shows that the development of molecular based techniques in detecetion and typing of C. difficile could be very valuable in combatting CDI. This also applies for the genetics and molecular biology of C. difficile in understanding the virulence of C. difficile. However, the continuing development of new techniques, both typing and genetics manipulation tools, has also led to the recognition of potential weaknesses of the currently frequent used techniques. Therefore, it is of utmost importance to keep up with the technical progress in the field of C. difficile research to enable further elucidation of the mechanisms that determine the virulence of C. difficile.